CN111876437B - Chimeric antigen receptor for targeting CD19 and interferon synergism and application thereof - Google Patents

Abstract

The invention relates to the field of chimeric antigen receptors, and discloses a chimeric antigen receptor for targeting CD19 and interferon synergy and application thereof, in particular to a polynucleotide sequence selected from: (1): the recombinant human IFN-beta polypeptide comprises a coding sequence of an anti-CD 19 single-chain antibody, a coding sequence of a human CD8 hinge transmembrane region, a coding sequence of a human 4-1BB intracellular region, a coding sequence of a human CD3 zeta intracellular region, a coding sequence of a human P2A peptide, a coding sequence of an EGFRT gene, a coding sequence of a human P2A peptide and a human IFN full-length sequence which are connected in sequence; and (2): the complement of the polynucleotide sequence of (1). The invention also discloses related fusion protein, nucleic acid construct, retrovirus and gene modified T cell, and application of the above substances in preparing medicine for treating CD19 mediated diseases.

Description

Chimeric antigen receptor for targeting CD19 and interferon synergy and application thereof

Technical Field

The invention relates to the field of chimeric antigen receptors, in particular to a chimeric antigen receptor which contains CD19-CAR and secretes interferon and application thereof.

Background

Chimeric antigen receptor T (CAR-T) cell therapy is one of the major breakthroughs of immune medicine in recent years. The CAR-T technology is based on the basic principle that T cells of a tumor patient are transformed into CAR-T cells capable of specifically attacking the tumor through genetic engineering, and then the CAR-T cells are returned to the patient body to ablate the tumor. The CAR-T technology was designed and reported by the israeli scientist Eshhar Zelig as early as 1993 and is continually optimized and refined over two decades thereafter. In the presence of antigen, T cells need to receive three signals in sequence to be fully activated and to proliferate and differentiate normally. The three signals are: a first signal, an antigen-bound T Cell Receptor (TCR), and a CD3 intracellular immunoreceptor tyrosine-activation motif (ITAM) transduction signal (CD 3 ζ); a second signal, a costimulatory signal, including surface receptors such as CD28, CD137, CD 134; a third signal, a cytokine, such as type I interferon, interleukin 12 (IL-12), and the like. The CAR-T design takes into account the immunological properties of T cell activation, and the structure of the CAR comprises an extracellular binding region, a transmembrane region, and an intracellular signaling region. Typically, the extracellular domain comprises a scFv capable of recognizing a tumor associated antigen, the transmembrane domain comprises a transmembrane domain of a molecule such as CD8 or CD28, and the intracellular signaling domain comprises an intracellular signaling domain of an Immunoreceptor Tyrosine Activation Motif (ITAM) CD3 ζ and a costimulatory signaling molecule such as CD28, CD137 or CD 134. The current CARs in the market are designed as second generation CARs, i.e. the CARs provide the first signal (ITAM domain) and the second signal (B7/CD 28 or 4-1BB/CD137 endodomain) necessary for T cell activation in the intracellular signaling region of the CAR, which can cause the sustained proliferation of T lymphocytes, increase the cytotoxicity, proliferative activity, etc. of T cells.

In 2010, the first B-cell lymphoma patient received CD 19-targeted CAR-T cell therapy and achieved exciting positive results. Since then, an increasing number of research groups have demonstrated the safety and efficacy of CAR-T technology in the treatment of B-cell hematological tumors through clinical trials. Although CAR-T cell therapy exhibits great advantages over traditional therapies in the area of hematological tumors, its problems remain elusive. For example, in some patients with less active T cells, CAR-T cells have less proliferative capacity and less efficient killing of tumor cells. In addition, most of the existing CAR-T generally has the defect of insufficient in-vivo continuous survival capability and is easy to cause tumor recurrence. However, the application dosage of the CAR-T cells is increased blindly, so that strong toxic and side effects such as inflammatory factor storm and central nervous system toxicity are easily caused. Thus, there remains an urgent need to engineer CAR designs to further improve CAR-T cell activity and proliferative capacity.

Disclosure of Invention

In order to further improve the killing effect of CAR-T on B cell lymphoma, the invention provides a chimeric antigen receptor comprising a CD19-CAR-EGFRT-IFN sequence and uses thereof. The invention improves on CD 19-targeted CAR design by adding an EGFRT suicide gene fragment and a gene-optimized human full-length Interferon (IFN) fragment to the C-terminus of a CD 19-CAR. CAR-T cells expressing CD19-CAR-EGFRt-IFN have greater tumor killing ability than CAR-T cells expressing CD19-CAR alone.

One idea to improve the anti-tumor effect of CAR-T is to increase cytokine expression at the tumor site. Cytokines can modulate the immune microenvironment around the tumor tissue while acting as a third signal, further increasing the level of CAR-T cell response. Cytokines include interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, growth factors, etc., and are many hundreds of species. In the previous research process, the applicant finds that any cytokine can not play a remarkable synergistic effect on T cells, and the invention selects the type I interferon as a third signal for the following reasons. Firstly, the applicant has conducted a great deal of research on type I interferon in the early period and has deeper and comprehensive understanding on the physiological function and potential side effect; secondly, type I interferons have multiple regulatory effects, on the one hand by directly inhibiting proliferation and regulating apoptosis, differentiation, migration and cell surface antigen expression of tumor cells, and on the other hand activating anti-tumor immunity, including: stimulating innate and adaptive cytotoxic lymphocytes such as T cells, NK cells, dendritic cells, innate Lymphocytes (ILCs) and immunosuppressive cells whose negative regulation blocks antitumor activity such as myeloid suppressor cells (MDSCs) and regulatory T (Treg) cells. In the prior art, interferon has a better treatment effect on malignant hematological tumors, such as Chronic Myelogenous Leukemia (CML), and IFN alpha 2b recombinant protein injection combined with chemotherapy or targeted therapy can effectively improve the survival time of patients. However, the direct injection of recombinant interferon protein has short half-life in vivo and is not easy to reach the focus, thus limiting the clinical application of the treatment method.

In a large number of previous studies, the applicant found that the expression of the full-length human IFN gene in tandem at the C-terminus of the CAR could significantly improve the level of CAR-T cell response. The design is exquisite in that the CAR gene and the IFN gene are separated by P2A peptide, so CAR and secreted IFN protein can be expressed simultaneously. When the CAR-T cell reaches the tumor focus and activates the CAR gene, the P2A peptide is hydrolyzed under the action of protease in the cell, and free IFN is released and secreted to the outside of the cell to play an immune activation function. The expression of IFN is regulated by CAR gene, so that the IFN activity can be released at the focus position, and the effect of precise synergy can be achieved.

The specific technical scheme of the invention is as follows:

first, the present invention discloses a polynucleotide sequence selected from the group consisting of:

(1): comprising the coding sequence of an anti-CD 19 single-chain antibody, the coding sequence of a human CD8 hinge transmembrane region, the coding sequence of a human 4-1BB intracellular region, the coding sequence of a human CD3 zeta intracellular region, the coding sequence of a human P2A peptide, the coding sequence of an EGFRT gene, the coding sequence of a human P2A peptide and the full-length sequence of human IFN which are connected in sequence, and

(2): the complement of the polynucleotide sequence of (1).

Preferably, the polynucleotide sequence further comprises a coding sequence of a signal peptide in front of the coding sequence of the anti-CD 19 single-chain antibody, and further preferably, the coding polynucleotide sequence of the signal peptide is shown as the 1 st-63 rd polynucleotide of SEQ ID NO. 1.

Preferably, the coding sequence of the anti-CD 19 single-chain antibody is shown in the 64 th-789 th polynucleotide of SEQ ID NO. 1.

Preferably, the coding sequence of the human CD8 hinge transmembrane region is shown as the polynucleotide in SEQ ID NO.1 from 790 to 996.

Preferably, the coding sequence of the human 4-1BB intracellular domain is as shown in the polynucleotide at positions 997-1137 of SEQ ID NO. 1.

Preferably, the coding sequence of intracellular domain of human CD3 zeta is as shown in the polynucleotide of SEQ ID NO.1 from 1138 to 1473.

Preferably, the coding sequence of the human P2A peptide is shown in SEQ ID NO 1 nucleotide sequences 1474-1551 and 2623-2700.

Preferably, the coding sequence of the EGFRT gene is shown as the 1618-2622 polynucleotide sequence in SEQ ID NO. 1.

Preferably, the polynucleotide sequence comprises a CSF2RA signal peptide at the N-terminus of the coding sequence of the EGFRt gene; the coding sequence of CSF2RA signal peptide is as SEQ ID NO.1, position 1552-1617 or amino acid sequence with similar or similar biological activity.

Preferably, the human IFN is human IFN α 2b, and the full-length sequence of the human IFN α 2b is a full-length cDNA sequence of the genetically optimized human IFN α 2b, referred to as oIFN α 2b. Further preferably, the coding sequence of oIFN alpha 2b is as shown in the 2701-3264 th polynucleotide of SEQ ID NO. 1.

After gene optimization, the expression efficiency of human IFN alpha 2b gene sequence can be enhanced by tens of times compared with the natural gene sequence.

Secondly, the present invention discloses a fusion protein selected from the group consisting of:

(1): a fusion protein comprising an anti-CD 19 single-chain antibody, a human CD8 hinge transmembrane region, a human 4-1BB intracellular region, a human CD3 zeta intracellular region, a human P2A peptide, EGFRT, a human P2A peptide and human IFN, which are linked in this order; and

(2): and (2) the fusion protein which is derived from the protein (1) and has similar or similar biological activity by substituting, deleting or adding one or more amino acids in the amino acid sequence defined in the protein (1).

Preferably, the fusion protein further comprises a CD8 signal peptide at the N-terminus of the anti-CD 19 single chain antibody. Further preferably, the amino acid sequence of the CD8 signal peptide is shown as SEQ ID NO. 2 amino acids 1-21 or an amino acid sequence with similar or similar biological activity.

Preferably, the amino acid sequence of the anti-CD 19 single-chain antibody is shown as amino acids 22-263 of SEQ ID NO. 2 or an amino acid sequence with similar or similar biological activity.

Preferably, the amino acid sequence of the human CD8 hinge transmembrane region is shown as the amino acid at the 264 th to 332 th positions of SEQ ID NO. 2 or an amino acid sequence with similar or similar biological activity.

Preferably, the amino acid sequence of the intracellular domain of human 4-1BB is as shown in amino acids 333-379 of SEQ ID NO. 2 or an amino acid sequence with similar or similar biological activity.

Preferably, the amino acid sequence of intracellular domain of human CD3 ζ is as shown in SEQ ID NO. 2 amino acids 380-491 or an amino acid sequence with similar or similar biological activity.

Preferably, the amino acid sequence of the human P2A peptide is shown as SEQ ID NO. 2 nucleotide sequences 492-517 and 875-900 or an amino acid sequence with similar or similar biological activity.

Preferably, the EGFRT amino acid sequence is SEQ ID NO. 2 amino acids 540-874 or an amino acid sequence with similar or similar biological activity.

Preferably, the fusion protein comprises a CSF2RA signal peptide at the N-terminus of the EGFRt; the amino acid sequence of CSF2RA signal peptide is 518-539 in SEQ ID NO. 2 or the amino acid sequence with similar or similar biological activity.

Preferably, the human IFN is human IFN alpha 2b protein, and the amino acid sequence is SEQ ID NO. 2 at positions 901-1088 or an amino acid sequence with similar or similar biological activity.

Third, the present invention discloses a nucleic acid construct comprising the polynucleotide sequence described above, or other polynucleotide sequences capable of encoding the fusion protein described above. Preferably, the nucleic acid construct is a vector. Further preferably, the nucleic acid construct is a retroviral vector comprising a replication initiation site, a 3 'LTR, a 5' LTR, a polynucleotide sequence as described hereinbefore, and optionally a selectable marker.

Fourth, the present invention discloses a retrovirus containing a nucleic acid construct as described hereinbefore, preferably containing said vector, more preferably containing said retroviral vector.

Fifth, the present invention discloses a method for transduction of retrovirus, which comprises small-scale packaging of the retrovirus, screening and establishing a virus-producing cell line, and large-scale transduction of T cells using the supernatant of the virus-producing cell line.

In a sixth aspect, the invention discloses a genetically modified T cell comprising a polynucleotide sequence as described above, or comprising a nucleic acid construct as described herein, or infected with a retrovirus as described herein, or stably expressing a fusion protein as described above.

Seventh, the present invention discloses the use of the genetically modified T cell as described above in the preparation of a medicament for the treatment of a CD19 mediated disease.

Preferably, the CD 19-mediated disease is B-cell lymphoma.

Compared with the prior art, the invention has the beneficial effects that:

the invention adopts the gene sequence of anti-CD 19 single-chain antibody, and searches the information of human CD8 hinge transmembrane region, human 4-1BB intracellular region, human CD3 zeta intracellular region, human P2A peptide and human IFN gene sequence (cDNA full-length sequence of human IFN alpha 2b gene) from NCBI GenBank database. The cDNA full-length sequence of the human IFN alpha 2b gene is subjected to gene optimization to obtain an IFN full-length sequence (oIFN alpha 2 b) with highest expression efficiency in human T cells.

The invention synthesizes the gene segment of the chimeric antigen receptor anti-CD 19 scFv-CD8 hinge transmembrane region-4-1 BB-CD3 zeta-EGFRT-oIFN alpha 2b by the whole gene and inserts the gene segment into a retrovirus vector. The recombinant plasmid packages the virus in ECO cells, infects T cells, and causes the T cells to express the chimeric antigen receptor. The transduction method of the present invention for modifying T lymphocytes with the chimeric antigen receptor gene is based on a retrovirus transduction method. The method has the advantages of high transduction efficiency, stable expression of exogenous genes, high batch stability, shortened time for in vitro culture of T lymphocytes to reach clinical level, and the like. The transduced nucleic acid is expressed on the surface of the CAR-T cell by transcription and translation. By detecting the expression of EGFR by flow cytometry, the proportion of retroviral-infected T lymphocytes and the expression of CAR on the cell surface can be calculated. The invention transduces T lymphocytes through retrovirus, and the proportion of the obtained CAR positive T lymphocytes is up to 70%. In vitro enzyme-linked immunosorbent assay (ELISA) detection shows that CAR-T cells can secrete a large amount of IFN to the culture supernatant, which indicates that retrovirus successfully transduces T cells and expresses secretory IFN. The killing function of CAR-T cells on specific tumor cells can be detected by Lactate Dehydrogenase (LDH) cytotoxicity detection assays. The CAR-T cells prepared by the invention have strong killing function on CD19 positive tumor cells, and the killing efficiency exceeds 80% under the condition that the effective target ratio is 3: 1. In addition, the present invention adds an EGFRt sequence in the CAR gene. The EGFRT gene segment expresses EGFR analogues with intracellular and extracellular signal transduction active fragments removed by genetic engineering, and only the fragment domain capable of responding to cetuximab is reserved. Therefore, EGFRT as an advanced suicide gene switch can realize complete controllability of CAR-T cell retention in vivo under the induction of monoclonal antibody drug cetuximab, thereby avoiding potential toxic and side effects caused by CAR-T cells to a certain extent.

The invention adds the human IFN full-length gene at the C-terminal of the CAR for the first time, and the CAR-T cell which simultaneously expresses the CAR and releases secretory human IFN protein is obtained. The research results in animals prove that the design of the CD19-CAR-EGFRT-IFN can obviously improve the tumor killing efficiency of CAR-T cells. Thus, the invention enhances the utility of CAR-T cells in CD19 mediated diseases.

Drawings

FIG. 1 is a schematic representation of the full length sequence of CD19-CAR-EGFRT-IFN α 2 b; an ScFv: a single chain antibody variable region; hinge: a CD8 hinge region; TM: the CD8 transmembrane domain.

FIG. 2 is a flow cytometry analysis showing CD4 after 3 days of retroviral infection of T cells ⁺ Subgroup and CD8 ⁺ CTR T, CTR CAR T, CD 19T and CD19 of the subgroups&The positive rate of EGFR on the surface of IFN α 2b T cells, i.e. the efficiency of CAR expression. CD 19T cells are T cells expressing CD19-CAR, CTR T cells are T cells not transduced with CAR, CTR CAR T cells are T cells expressing non-CD 19A target control CAR-T cell.

FIG. 3 shows the content of IFN α 2b in the supernatants of CD19& IFN α 2b T, CD19T and CTR CAR T cell culture media after enzyme-linked immunosorbent assay (ELISA) detection of retroviral infection.

FIG. 4 shows the LDH assay for the target cell lysis rate after coculture of CAR-T cells and target cells at different effective target ratios.

FIG. 5 is a graph of D-luciferin sodium salt imaging after tail vein injection of CAR-T cells in tumor transplantation models, and observation of tumor cell residues in mice at different time points. A, main experimental process; b, counting the fluorescein intensity in the mice of each group at different time points; c, pictures show sodium salt imaging results of mice of each group.

Detailed Description

The present invention will be further described with reference to the following examples.

The present invention provides a fusion protein comprising a CD 19-targeted Chimeric Antigen Receptor (CAR). The fusion protein comprises an anti-CD 19 single-chain antibody, a human CD8 hinge transmembrane region, a human 4-1BB intracellular region, a human CD3 zeta intracellular region, a human P2A peptide and a human IFN full-length fragment which are connected in sequence.

The present invention includes polynucleotide sequences encoding the fusion proteins of the present invention. The polynucleotide sequences of the invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

The coding sequence of the human IFN full-length fragment is a human IFN gene cDNA full-length sequence subjected to gene optimization. It is understood that gene optimization, also known as codon optimization, refers to the replacement of one or more nucleotides in a polynucleotide sequence encoding a protein without altering the amino acid sequence of the protein, in order to increase the expression level and efficiency of the protein in cells of a particular species. The gene optimization includes but is not limited to methods of codon preference optimization, RNA advanced structure optimization, enzyme cutting site optimization, GC content adjustment and the like. The present invention includes various human IFN encoding polynucleotide sequences (oIFN) obtained by the above gene optimization method. The common feature of these polynucleotide sequences is the use of different nucleotide codons, but the encoded amino acid sequence is identical to the full-length cDNA sequence of wild-type human IFN. Sequence identity between two aligned polynucleotide sequences and between amino acid sequences can be calculated using, for example, BLAST and BLASTp from NCBI. As an illustrative example, the coding sequence of oIFN α 2b of the present invention is shown in the 2701-3264 th polynucleotide of SEQ ID NO. 1.

anti-CD 19 single chain antibodies suitable for use in the present invention may be derived from a variety of anti-CD 19 monoclonal antibodies known in the art. The basic structure of a single chain antibody comprises a light chain variable region, a linker sequence and a heavy chain variable region. Preferably, the light chain variable region is of the kappa chain type. As an illustrative example, the amino acid sequence of the variable region of the light chain of the anti-CD 19 single-chain antibody of the present invention is shown as amino acids 22-128 of SEQ ID NO 2. As an illustrative example, the amino acid sequence of the heavy chain variable region of the anti-CD 19 single-chain antibody of the present invention is shown as amino acids 144 to 263 of SEQ ID NO 2. The variable region of the light chain and the variable region of the heavy chain of the single-chain antibody are connected by a linker sequence. The linker sequence may be one known in the art to be suitable for use with antibodies, for example, a G and S containing linker sequence. Typically, the linker contains one or more motifs which repeat back and forth. Preferably, the motifs may be GGGS, GGGGS, SSSSSG, GSGSA and GGSGG, and the linker comprises from 1 to 5 repeat motifs with no intervening amino acid residues between adjacent repeat motifs. As an illustrative example, the variable region of the light chain and the variable region of the heavy chain of the anti-CD 19 single-chain antibody of the present invention are linked by (GGGGS) 3, and the amino acid sequence of the linker sequence is shown as amino acids 129-143 of SEQ ID NO. 2.

Human CD8 hinge transmembrane regions suitable for use in the present invention can be the various human CD8 hinge transmembrane region sequences commonly used in the art for CARs. As an illustrative example, the amino acid sequence of the human CD 8. Alpha. Hinge transmembrane region of the present invention is shown as amino acids 264 to 332 of SEQ ID NO 2.

The human 4-1BB intracellular domain suitable for use in the present invention may be any of the various human 4-1BB intracellular domains known in the art for CAR. As an illustrative example, the amino acid sequence of the intracellular domain of human 4-1BB for use in the present invention is shown in positions 333 to 379 of SEQ ID NO. 2.

The human CD3 ζ intracellular region suitable for use in the present invention may be various human CD3 ζ intracellular regions conventionally used in the art for CARs. As an illustrative example, the amino acid sequence of the intracellular domain of human CD3 ζ is shown as amino acids 380 to 491 of SEQ ID NO 2.

Human P2A peptides suitable for use in the invention can be various self-cleaving sequences conventionally used in the art for CARs. As an illustrative example, the amino acid sequence of the human P2A peptide is shown as amino acids 492-517 and 875-900 of SEQ ID NO 2.

Suitable signal peptides for use in the present invention are the CD8 signal peptide and the CSF2RA signal peptide, as well as signal peptides conventionally used in the art to localize exogenously expressed recombinant proteins to the cell membrane. As an illustrative example, the amino acid sequence of the CD8 signal peptide is shown as amino acids 1-21 of SEQ ID NO. 2. As an illustrative example, the amino acid sequence of the CSF2RA signal peptide is shown as amino acids 518-539 of SEQ ID NO. 2.

The invention also includes a CAR represented by the amino acid sequence at positions 22-491 of SEQ ID NO. 2, a CAR represented by the amino acid sequence at positions 22-874 of SEQ ID NO. 2, a CAR represented by the amino acid sequence at positions 22-1088 of SEQ ID NO. 2, a CAR represented by the amino acid sequence at positions 1-491 of SEQ ID NO. 2, a CAR represented by the amino acid sequence at positions 1-874 of SEQ ID NO. 2, or a mutant of a CAR represented by SEQ ID NO. 2. These mutants include: an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity to the CAR and retaining the biological activity of the CAR (e.g., activating T cells). Sequence identity between two aligned sequences can be calculated using, for example, BLASTp by NCBI.

The mutants also include: the amino acid sequence shown in positions 22-491 of SEQ ID NO 2, the amino acid sequence shown in positions 22-874 of SEQ ID NO 2, the amino acid sequence shown in positions 22-1088 of SEQ ID NO 2, the amino acid sequence shown in positions 1-491 of SEQ ID NO 2, the amino acid sequence shown in positions 1-874 of SEQ ID NO 2 or the amino acid sequence with one or several mutations (insertions, deletions or substitutions) in SEQ ID NO 2, while still retaining the biological activity of the CAR. The number of mutations usually means within 1-10, such as 1-8, 1-5 or 1-3. The substitution is preferably a conservative substitution. For example, conservative substitutions with amino acids of similar or similar properties are not typically used in the art to alter the function of a protein or polypeptide. "amino acids with similar or analogous properties" include, for example, families of amino acid residues with analogous side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, substitution of one or more sites with another amino acid residue from the same side chain species in a polypeptide of the invention will not substantially affect its activity.

The present invention uses the gene sequence of an anti-CD 19 single-chain antibody (specifically, scFV derived from clone No. C11D5.3), and searches the NCBI GenBank database for information on the human CD8 hinge transmembrane region, the human 4-1BB intracellular region, the human CD3 zeta intracellular region, the human P2A peptide, and the human IFN gene sequence (the full-length cDNA sequence of human IFN. Alpha.2b). The human IFN gene sequence is subjected to gene optimization to obtain an IFN full-length sequence (oIFN alpha 2 b) with the highest expression efficiency in human T cells.

The invention synthesizes the gene segment of the chimeric antigen receptor anti-CD 19 scFv-CD8 hinge transmembrane region-4-1 BB-CD3 zeta-oIFN through the whole gene and inserts the gene segment into a retrovirus vector. The recombinant plasmid packages the virus in ECO cells, infects T cells, and makes the T cells express the chimeric antigen receptor. The transduction method of the present invention for genetically modifying T lymphocytes with a chimeric antigen receptor is based on a retroviral transduction method. The method has the advantages of high transduction efficiency, stable expression of exogenous genes, high batch stability, shortened time for in vitro culture of T lymphocytes to reach clinical level, and the like. The transduced nucleic acid is expressed on the surface of the CAR-T cell by transcription and translation. The proportion of retrovirus-infected T lymphocytes and the expression of CAR on the cell surface can be calculated by detecting the content of EGFRT co-expressed with CAR gene by EGFR antibody using flow cytometry. The invention transduces T lymphocytes through retrovirus, and the proportion of the obtained CAR positive T lymphocytes is up to 70%. In vitro enzyme-linked immunosorbent assay (ELISA) detection shows that the CAR-T cells can secrete a large amount of IFN protein to the culture supernatant, which indicates that retrovirus successfully transduces the T cells and expresses the secretory IFN protein. The killing function of CAR-T cells on specific tumor cells can be detected by a Lactate Dehydrogenase (LDH) cytotoxicity detection assay. The CAR-T cells prepared by the invention have strong killing function on CD19 positive tumor cells, and the killing efficiency exceeds 80% under the condition that the effective target ratio is 3: 1.

The invention obtains the CD19-CAR-EGFRt-IFN polynucleotide sequence by adding a gene optimized human IFN full-length coding sequence at the C-terminal end of the CD19-CAR polynucleotide sequence. Wherein the IFN gene sequence can be the full-length sequence of any gene among human IFN alpha 2a, human IFN alpha 2b and human IFN beta. The human IFN alpha 2a and human IFN alpha 2b amino acid sequence is highly similar, only in the 23 th amino acid difference (human IFN alpha 2a 23 rd amino acid is K, human IFN alpha 2b 23 rd amino acid is R, belonging to conservative substitution). Human IFN beta also belongs to type I interferon, has similar biological functions compared with the two types I interferon, and has the functions of inducing tumor cell apoptosis and regulating immune cell activity. Often in clinical research IFN alpha 2 and IFN beta mutual replacement use. Animal experiments show that after the C-terminal of the CD19-CAR is added with a full-length fragment of a human IFN alpha 2b gene, the CAR-T cells expressing the CD19-CAR-EGFRT-IFN alpha 2b have stronger tumor killing capability in animals compared with the CD19-CAR sequence. The applicants therefore believe that any of the three genes described above can act synergistically on CAR-T cells.

The invention is described in further detail below by way of a series of experimental examples, taking the CD19-CAR-EGFRT-IFN α 2b design as an example. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should in no way be construed as limited to the following examples, but rather should be construed to include any and all variations which become apparent in light of the teachings provided herein. The methods and reagents used in the examples are, unless otherwise indicated, conventional in the art.

Example 1: determination of CD19-CAR-EGFRT-IFN alpha 2b gene sequence and construction of retroviral vector

From NCBI website database search for human CD8 hinge transmembrane region, human 4-1BB intracellular region, human CD3 zeta intracellular region, EGFRT and human IFN alpha 2b full-length cDNA sequence information. The full-length cDNA sequence of the wild-type human IFN alpha 2b gene is called nIFN alpha 2b. Codon optimization is carried out on the nIFN alpha 2b sequence on website http:// sg.idtdna.com/site to obtain oIFN alpha 2b, and the better suitability for human cell expression under the condition of unchanged coding amino acid sequence is ensured.

The full-length polynucleotide sequence of CD19-CAR-EGFRT-IFN alpha 2b is obtained according to the sequence of CD19 scFv, human CD8 hinge transmembrane region, human 4-1BB intracellular region, human CD3 zeta intracellular region, human P2A peptide, EGFRT, human P2A peptide and oIFN alpha 2b. Simultaneously constructing a CD19-CAR-EGFRT full-length polynucleotide sequence only comprising a CD19 scFv, a human CD8 hinge transmembrane region, a human 4-1BB intracellular region, a human CD3 zeta intracellular region, a human P2A peptide and EGFRT. The sequence information of the full-length polynucleotide of the CD19-CAR-EGFRT-IFN alpha 2b is shown in a nucleotide sequence table (SEQ ID NO. 1). The full-length polynucleotide sequence of the CD19-CAR-EGFRT is shown as the polynucleotide at the 1 st-874 th site of SEQ ID NO. 1. All of the above polynucleotides were synthesized in Ongchow Biotech, inc., cloned in pUC57 vector, and sequenced again.

The nucleotide sequence of CAR-EGFRT-IFN alpha 2b was double-digested with NotI (NEB) and EcoRI (NEB), ligated by T4 ligase (NEB), inserted into the NotI-EcoRI site of retrovirus (MP 71), and transformed into competent E.coli (DH 5. Alpha.).

The plasmid was extracted and purified using a plasmid purification kit from Qiagen, and the resulting CD19-CAR-EGFRT-IFN α 2b plasmid was used for retroviral packaging experiments.

The CD19-CAR-EGFRT sequence was inserted into the retroviral vector in the same manner as described above to construct a retroviral vector containing the CD19-CAR-EGFRT sequence. And extracting plasmids for retrovirus packaging.

The plasmid map of CD19-CAR-EGFRT-IFN alpha 2b constructed in the example is shown in FIG. 1.

Example 2: establishment of retroviral packaging and toxigenic strains

Using the retroviral vectors prepared in example 1 containing CD19-CAR-EGFRt-IFN α 2b and CD19-CAR-EGFRt, two retroviruses were packaged separately as follows:

1. day 1: phoenix Ecotropic (ECO) cells should be less than 20 passages, but not overgrown. At 0.6X 10 ⁶ Laying the cells in a density plate of per ml, adding 10ml of DMEM medium into a 10cm dish, fully and uniformly mixing the cells, and culturing the cells at 37 ℃ overnight;

2. day 2: the ECO cell fusion degree reaches about 90 percent for transfection (usually, the plating lasts for about 14-18 h); preparation of plasmid MP 71-target Gene 12.5. Mu.g, 1.25M CaCl ₂ 250μl，H ₂ O1 ml, the total volume is 1.25ml; the other tube was filled with an equal volume of 2 × HBS to the plasmid complex and vortexed for 20s while adding the plasmid complex. The mixture was gently added to the ECO dish edge to edge, incubated at 37 ℃ for 4h, medium removed, washed once with PBS, and re-added with pre-warmed fresh medium.

3. Day 4: after transfection for 48h, the supernatant was collected and filtered through a 0.45um filter to obtain a retrovirus solution, which was stored at-80 ℃.

4. Establishing an toxigenic strain: the obtained retrovirus infects HY268 cells, and after two days of infection, flow cell sorting is carried out, and the cell strain with the highest secretory retrovirus titer and derived from single cells is screened and stored for a long time. Retroviral supernatant can be prepared in large scale by using the cell strain for preparing CAR-T cells by gene transduction.

Example 3: retroviral infection of human T cells

1. Resuscitating frozen healthy human peripheral blood PBMC, adjusting cell density to 1-2 × 10 with 10% FBS-containing RPMI-1640 complete medium ⁶ /ml。

2. Collecting PBMC and magnetic beads from Ficoll separating medium (tertiary sea level)Separating to obtain relatively pure CD3 ⁺ T cells, magnetic beads CD3 ⁺ The cell ratio was 3.

3. The day after T cell activation, non-tissue treated plates were coated with Retronectin (Takara) diluted with PBS to a final concentration of 15. Mu.g/ml, 1.2 ml per well of 6-well plates. Protected from light and kept at 4 ℃ overnight for use.

4. After two days of T cell activation culture, the coated 6-well plate was taken out, the coating solution was aspirated away, and the plate was washed once with PBS.

5. The retrovirus solution prepared in example 2 was added to each well at a temperature of 5 to 6ml,32 ℃ and 2000 Xg, and centrifuged for 2 hours. 3ml of fresh complete medium containing hIL-2 (100U/ml) was added to each well and incubation was continued for 1 day.

6. After the cells are infected, the density of the cells is observed every day, and the culture solution of the T cells containing IL-2 100U/ml is supplemented at appropriate time to maintain the density of the T cells at 5X 10 ⁵ About/ml, which is convenient for cell expansion.

7. CAR-T cells infected with the two retroviruses prepared in example 2, respectively, were thus obtained, named CD19& IFN α 2b T cells (expressing CD19-CAR-EGFRt-IFN α 2b of example 1) and CD 19T cells (expressing CD19-CAR-EGFRt of example 1), respectively.

8. A Control group not infected with virus was set, and a retrovirus solution was replaced with an equal volume of PBS solution, and Control T (CTR T) cells were obtained in the same manner as described above.

9. And (3) setting a CAR-T Control group with a non-CD 19 target, replacing CD19-CAR-EGFRt-IFN alpha 2b with a CAR with a non-CD 19 target, and obtaining a Control CAR T (CTR CAR T) cell according to the same method.

Example 4: proportion of CAR-positive T lymphocytes after infection, expression of surface CAR protein and detection of IFN alpha 2 content in supernatant

We used FACS methods to demonstrate the proportion of CAR positive T lymphocytes and the expression of CAR proteins by detecting EGFR expression. Simultaneously, the IFN alpha 2 content in the cell culture supernatant is detected by enzyme-linked immunosorbent assay (ELISA).

FIG. 2 shows, using an example3 day 3 after infection of T cells with the prepared retrovirus, CD4 ⁺ The positive rate of EGFR (CAR) in both T cells and CD8+ T cells reached 80%.

Two CAR-T cells, CTR T cells and CTR CAR T cells prepared in example 3 72 hours after infection (control group) were collected by centrifugation, respectively, and the supernatant after culture was collected. ELISA test the supernatant IFN alpha 2 content.

FIG. 3 shows ELISA results, CD19& IFN alpha 2b T cell supernatant IFN alpha 2 content significantly higher than CTR T and CD 19T cells. The results confirmed that the CD19& IFN alpha 2b T cells can express secreted IFN alpha 2.

Example 5: detection of tumor specific cell killing effect by Lactate Dehydrogenase (LDH) method

1. Adjusting the concentration of target cells (TX 858) to 4X 10 ⁵ Each 50 μ l of target cells and effector cells (effective to target ratio 3, 1. The effector cells are CTR CAR T cells, CD 19T cells and CD19, respectively&IFN alpha 2b T cells. In addition, a target cell natural release hole, an effector cell natural release hole and a target cell maximum release hole were provided, and 50. Mu.l each of the target cells and the culture solution was added. All the above-mentioned items are equipped with three complex holes.

2. The cells were incubated at 37 ℃ with 5% CO ₂ Culturing in an incubator for 4 h.

3. Cell culture was terminated 45 min before cell culture, and 10. Mu.l of lysis solution was added to the maximum release pore of the target cell.

The 4.96-well plate was centrifuged at 1500 rpm/min for 5 min, 50. Mu.l of supernatant was pipetted per well and placed in a flat-bottomed 96-well plate, and 50. Mu.l of LDH substrate was added simultaneously, and the reaction was carried out for 30 min at room temperature in the dark.

5. The wells were terminated by adding 50. Mu.l of 1mol/L acetic acid solution, and the optical density value (A490) was measured at 490nm of a microplate reader, and two-wavelength measurement was conducted using the wavelength of 630 nm as a reference wavelength.

% cytotoxicity rate = (experimental group-effector cell self-release group-target cell self-release group) × 100/(target cell maximum release group-target cell self-release group)

Figure 4 shows that target cell lysis rates were measured by the LDH method after co-culture using CD19& IFN α 2b T cells and target cells TX858 at different effective target ratios of 3, 1. The result shows that when the effective target ratio is 3:1, the cell lysis rate reaches more than 80 percent; when the effective target ratio is 1 to 3, the cell lysis rate is still about 20%.

Example 6: detection of tumor killing effect of CAR-T cells in animal body by tumor transplantation model

1. The tail vein of a B-NDG severe combined immunodeficiency mouse (Baiosacchus) is inoculated with human lymphoma cells Raji-Luc with fluorescein labels. The inoculation amount is 1 multiplied by 10 ⁶ 0.3ml. Randomized into 3 experimental groups, a T cell control group (CTR CAR T) infected with a non-CD 19 replacement CAR virus, a CD 19T cell control group, and CD19&IFN alpha 2b T cell group, each group of 6 mice.

2.5 days after tumor cell inoculation, different types of CAR-T cells were injected into tail vein of mice respectively, and the injected amount of CAR-T cells was 5X 10 ⁶ CAR ⁺ T/0.2 ml。

3. Sodium salt imaging was performed by intraperitoneal injection of 3mg of D-luciferin into mice 7, 14, 21 and 28 days after CAR-T cell injection, respectively. The number of residual tumor cells in the mice was observed, and the fluorescein intensity (photon density) was counted.

Figure 5 shows a significant reduction in human lymphoma cell residual in mice injected with CD 19T and CD19& IFN α 2b T compared to the CTR CAR T control group. Of particular note, CD 19T does not kill minute tumor cell residues, which grow rapidly as clones after 28 days, forming new tumors, a phenomenon that essentially mimics the phenotype of tumor recurrence after CAR-T treatment in clinical studies. In contrast, the effect of CD19& IFN α 2b T cells on tumor killing was better, suggesting that CD19& IFN α 2b T may play an important role in preventing tumor recurrence.

The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.

Sequence listing

<110> Zhejiang Kangbaiyu Biotechnology Co., ltd

<120> chimeric antigen receptor for targeting CD19 and interferon synergy and application thereof

<130> 2019

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3264

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60

cctgacattc agatgactca gaccacaagc agcctcagtg cgagcctggg ggacagggtg 120

actatcagct gccgggccag ccaggacatt tccaagtacc tgaattggta ccagcagaag 180

cccgatggta ctgtgaaact cctgatatat catacttcta ggctccattc cggggttcca 240

agccgattca gtggctccgg ttccggtaca gattattccc tgaccattag caacttggaa 300

caggaggaca ttgcaacgta tttctgtcag caaggcaaca cattgcccta cacattcggg 360

ggcgggacta aactcgaaat aactggcggc gggggttctg gtggcggcgg cagcggcggt 420

ggaggatcag aagtgaagct gcaggaaagt ggccccgggc tggtagcccc aagtcagtcc 480

ctgagtgtaa cctgtacagt gagtggagtg tctcttcctg actacggggt aagttggatt 540

cggcaacctc cacgcaaggg cctggagtgg ctcggcgtga tttggggatc tgagacaact 600

tactacaatt ccgccctgaa gagcaggctg accatcatta aggacaatag caagtcacag 660

gtgtttctga agatgaactc actgcagacc gacgacaccg ccatctatta ctgcgccaaa 720

cattattatt atggcgggag ttatgctatg gactactggg gccagggcac tagcgtcacc 780

gtcagcagta ctacaactcc agcacccaga ccccctacac ctgctccaac tatcgcaagt 840

cagcccctgt cactgcgccc tgaagcctgt cgccctgctg ccgggggagc tgtgcatact 900

cggggactgg actttgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960

gtccttctcc tgtcactggt tatcaccctt tactgcaggt tcagtgtcgt gaagagaggc 1020

cggaagaagc tgctgtacat cttcaagcag cctttcatga ggcccgtgca gactacccag 1080

gaggaagatg gatgcagctg tagattccct gaagaggagg aaggaggctg tgagctgaga 1140

gtgaagttct cccgaagcgc agatgcccca gcctatcagc agggacagaa tcagctgtac 1200

aacgagctga acctgggaag acgggaggaa tacgatgtgc tggacaaaag gcggggcaga 1260

gatcctgaga tgggcggcaa accaagacgg aagaaccccc aggaaggtct gtataatgag 1320

ctgcagaaag acaagatggc tgaggcctac tcagaaatcg ggatgaaggg cgaaagaagg 1380

agaggaaaag gccacgacgg actgtaccag gggctgagta cagcaacaaa agacacctat 1440

gacgctctgc acatgcaggc tctgccacca agacgagcta aacgaggctc aggcgcgacg 1500

aactttagtt tgctgaagca agctggggat gtagaggaaa atccgggtcc catgttgctc 1560

cttgtgacga gcctcctgct ctgcgagctg ccccatccag ccttcctcct catcccgcgg 1620

aaggtgtgca atggcatagg cattggcgag tttaaagatt ctctgagcat aaatgctacg 1680

aatattaagc atttcaagaa ttgtacttct attagtggcg acctccatat tcttccggtt 1740

gccttcaggg gtgactcttt cacccacaca cctccattgg atccacaaga acttgacatc 1800

ctgaagacgg ttaaagagat tacaggcttc ctccttatcc aagcgtggcc cgagaacaga 1860

acggacttgc acgcctttga gaacctcgaa ataatacggg gtcggacgaa gcaacacggc 1920

caatttagcc ttgcggttgt tagtctgaac attacttctc tcggccttcg ctctttgaaa 1980

gaaatcagcg acggagatgt catcattagt ggaaacaaga acctgtgcta cgcgaacaca 2040

atcaactgga agaagctctt cggtacttca ggccaaaaga caaagattat tagtaacaga 2100

ggagagaata gctgtaaggc taccggacaa gtttgtcacg ccttgtgtag tccagagggt 2160

tgctggggac cggaaccaag ggattgcgtc agttgccgga acgtgagtcg cggacgcgag 2220

tgtgtggata agtgcaatct tctggaaggg gaaccgcgag agtttgtaga aaattccgaa 2280

tgtatacagt gtcatcccga gtgtcttcca caagcaatga atatcacatg tacagggagg 2340

ggtcctgata actgtatcca atgtgcacac tacatagatg gtcctcactg tgtaaagacg 2400

tgccccgccg gagtaatggg tgaaaacaac accctcgtgt ggaagtacgc cgatgccggg 2460

catgtctgtc atttgtgtca tcccaactgc acatatggct gtaccggtcc tggattggag 2520

ggctgtccaa caaacgggcc gaaaataccg agtatcgcaa caggcatggt gggagcactt 2580

ttgcttctcc tcgttgtcgc cctgggcatc ggcttgttca tgcgagctaa acgaggctca 2640

ggcgcgacga actttagttt gctgaagcaa gctggggatg tagaggaaaa tccgggtccc 2700

atggccctga ccttcgccct gctggtggcc ctgctggtcc tgagctgcaa gagctcctgc 2760

agcgtggggt gcgacctgcc ccagacccac agcctgggct ccagaagaac cctgatgctg 2820

ctggcccaga tgagaagaat cagtctgttc agctgcctga aagacagaca cgactttggc 2880

ttccctcagg aggaatttgg aaaccagttc cagaaggccg aaaccatccc cgtgctgcac 2940

gagatgatcc agcagatctt caacctgttc tccaccaaag atagcagcgc agcctgggac 3000

gaaaccctgc tggacaagtt ctacaccgag ctgtaccagc agctgaacga cctggaggcc 3060

tgcgtgatcc agggcgtggg agtgaccgag acaccactga tgaaagagga tagcattctg 3120

gccgtgagga aatacttcca gagaatcacc ctgtacctga aagagaaaaa gtacagtccc 3180

tgcgcctggg aggtggtgag agccgagatc atgagaagct tcagcctgag caccaatctg 3240

caggaaagcc tgagaagcaa ggag 3264

<210> 2

<211> 1088

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Phe Ser Val

325 330 335

Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Arg Ala Lys Arg Gly

485 490 495

Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu

500 505 510

Glu Asn Pro Gly Pro Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys

515 520 525

Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn

530 535 540

Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr

545 550 555 560

Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His

565 570 575

Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro

580 585 590

Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr

595 600 605

Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His

610 615 620

Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly

625 630 635 640

Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu

645 650 655

Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn

660 665 670

Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly

675 680 685

Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser

690 695 700

Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly

705 710 715 720

Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser

725 730 735

Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro

740 745 750

Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys

755 760 765

Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn

770 775 780

Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr

785 790 795 800

Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr

805 810 815

Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr

820 825 830

Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys

835 840 845

Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu

850 855 860

Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Ala Lys Arg Gly Ser

865 870 875 880

Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu

885 890 895

Asn Pro Gly Pro Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu

900 905 910

Val Leu Ser Cys Lys Ser Ser Cys Ser Val Gly Cys Asp Leu Pro Gln

915 920 925

Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met

930 935 940

Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly

945 950 955 960

Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile

965 970 975

Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr

980 985 990

Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr

995 1000 1005

Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln

1010 1015 1020

Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu

1025 1030 1035 1040

Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys

1045 1050 1055

Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg

1060 1065 1070

Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu

1075 1080 1085

Claims (11)

1. A fusion protein, characterized in that: the amino acid sequence is shown as SEQ ID NO. 2.

2. A polynucleotide encoding the fusion protein of claim 1.

3. The polynucleotide of claim 2, wherein: the nucleotide sequence is shown as SEQ ID NO. 1.

4. A nucleic acid construct, comprising: a polynucleotide comprising a polynucleotide encoding the fusion protein of claim 1.

5. The nucleic acid construct of claim 4, wherein: the nucleotide sequence of the polynucleotide is shown in SEQ ID NO. 1.

6. The nucleic acid construct of claim 4, wherein: the nucleic acid construct is a vector.

7. The nucleic acid construct of claim 6, wherein: the nucleic acid construct is a retrovirus vector, and comprises a replication initiation site, 3 'LTR, 5' LTR, polynucleotide with a nucleotide sequence shown as SEQ ID NO.1, and a marker.

8. A retrovirus, comprising: comprising the nucleic acid construct of any of claims 4-7.

9. A method of transduction of a retrovirus, comprising: the transduction method includes a method of packaging the retrovirus of claim 8 on a small scale, a method of screening and establishing a virus-producing cell line.

10. A genetically modified T cell, characterized by: the T cell comprising the polynucleotide of claim 2 or 3, or comprising the nucleic acid construct of any one of claims 4 to 7, or infected with the retrovirus of claim 8, or stably expressing the fusion protein of claim 1.

11. Use of the genetically modified T cell of claim 10 in the manufacture of a medicament for treating a CD19 mediated B cell lymphoma disorder.

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